The present invention relates to a method and an apparatus for efficient proliferation and culture of adhesive cells followed by transfer of said cells into a mass culture tank so as to produce a physiologically active substance.
Culture of adhesive cells has conventionally been performed by use of various culture system as follow:
(a) So-called liquid tight system generally comprising a plurality of plates arranged in a parallel manner within a container in order to obtain a sufficiently large surface for cell adhesion. Upon completion of said cell adhesion onto the plates, a quantity of culture fluid is circulated in the container under control of pH and DO (dissolved oxygen). The so-called rotary system generally comprising a plurality of discs or the like also arranged in parallel within a cylinder set upright to achieve the cell adhesion onto the discs and then laid down to be rotated.
(b) A system comprising scrolled plastic film charged within a cylinder, wherein cell culture is performed in the same manner as the so-called roller culture. Then a quantity of culture fluid is circulated in the cylinder, set upright, for example, by air supplied into the cylinder.
(c) The multitray system of box-nest construction similar to the system set forth above in (a) except that there is gaseous phase and the culture can be stationary as achieved in roller botter, without the requirement for the culture fluid circulation.
(d) The plastic bag system comprising an oxygen or carbon dioxide-permeable plastic bag rolled up like the fire hose through which a quantity of culture fluid flows. This system facilitates the control of DO and pH.
(e) The hollow fiber system comprising hollow fibers as usually used for artificial kidney dialysis, wherein adhesion and proliferation of cells occur on the exterior side while nutrient supply occurs from the interior side of each hollow fiber, i.e., from the near side of the cell layer.
(f) The glass beads filled column system in which adhesion and proliferation of cells occur on the surfaces of glass beads charged within a container under circulation of a culture fluid having pH and DO previously adjusted.
(g) The microcarrier system utilizing, instead of said glass beads, minute beads of such specific gravity that these minute beads float in culture fluid under a gentle agitation in order of 20 to 40 r.p.m. and the culture, as well as proliferation of cells occur on their surfaces.
The systems (a) through (d) are exclusively for the batch production and the number of cells which can be cultivated for each process results in a poor yield of the target substance.
The systems (e) through (f), of the continuous culture medium circulation type, are also restricted in the number of cells which can be cultivated and is not suitable for mass culture.
Presently, the mass culture has mostly relied on the microcarrier system set forth above in (g) and such system having a capacity of 8000 is known.
One example of the microcarrier system is disclosed in Japanese Disclosure Gazette No. 0982-5670, which aims at efficient cell culture within a culture tank containing a cylindrical member set upright therein, by providing said cylindrical member therein with deflectors and connecting said cylindrical member with a culture fluid outlet conduit so that a desired quantity of culture fluid may be stably circulated for a long period. As a similar example, Japanese Disclosure Gazette No. 1985-168379 discloses a cell cultivating apparatus having a unit comprising a plurality of hollow fibers each having a wall-membrane which is cell-impermeable but nutrient-permeable, said unit being provided at opposite ends or one end with an inlet and an outlet for cell culture fluid so that high mass and high density culture can be achieved by suspension culture. As still another example, Disclosure Gazette No. 1985-259179 discloses a similar mass and high density cell culture tank of the suspension type which is provided at the top with an inlet for fresh culture fluid, at the bottom with an outlet for used culture fluid, and adjacent the top with an impeller.
The above-mentioned systems of the prior art are disadvantageous in that the cultivating capacity can be improved only by making the culture tank volume correspondingly larger and proliferation of cells from the initial stage in such larger culture tank would not only encounter additional problems as those in circulation and control of correspondingly increased culture fluid but also take a longer time period.
Particularly when it is desired to produce physiologically active substance by cell culture, the culture medium for cell proliferation has ingredients different from those for cell culture and the former becomes wasteful especially in the larger culture tank.
Accordingly, the method according to the present invention, in view of such problems encountered by the microcarrier system of well known art, intends to achieve an efficient cell culture by performing the initial cell proliferation and the subsequent cell culture for production of the physiologically active substance in different tanks.
For the cells which are readily subject to damage due to the shearing force and have relatively low colony formation efficiency it is very difficult to perform the microcarrier culture. To reduce the effect of said shearing force, the method is preferably, one in which the cells themselves are adhesively fixed onto particular material having a larger surface area in the stationary culture method and the culture medium is circulated, therethrough.
The previously mentioned hollow fiber system is one embodiment of such method. Specifically, the culture medium is continuously circulated not only through the interior but also along the exterior surface of the hollow fiber on which the adhesive cells are to be prolifirated so that the nutrient and metabolism product are efficiently moved between the interior and the exterior of the fiber, enabling the cells to be cultivated at a high density.
However, this system unacceptably complicates the apparatus and has not been commercially adopted for mass production.
Japanese Disclosure Gazette No. 1984-154984 discloses a simplified hollow fiber system in which the cells are proliferated, cultivated on ceramic matrix and the culture fluid is continuously circulated.
According to this prior art, alumina, silica, titanium, zircon or the like or combination thereof is sistered to form porous ceramic carrier which is a cylindrical monolithic carrier having at least about 20 through-holes extending in parallel to one another per square inch of cross-section. However, this system is inconvenient in that the carrier is readily clogged as it is continuously used.
As will be appreciated from the foregoing description, all of the conventional system are disadvantageous for mass production in commercial scale. To over come such problems, the inventors disclosed a solution in Japanese Disclosure Gazette No. 1987-236480. This solution is a method comprising steps of providing ceramic particles consisting for the most part of alumina suitable for all adhesion and having an approximately uniform size of 5 to 9 meshes, supplying culture fluid into a column filled with said ceramic particles, exchanging the quantity of aged culture fluid, after movement through the first half of the column, due to proliferation of cells with quantity of fresh culture fluid at an intermediate level of column, and removal of said aged culture is further continued through the second half of the column.
This method is characterized by that the effort of culture fluid to the cells is relatively uniform, the cells are free from the damage due to a shearing force and the carrier facilitates the cell adhesion, and thus the method is suitable for mass culture of adhesive cells.
Nevertheless, there still remains an important problem that, when cultivation is performed within a column or tank filled with granular sedimentary immobilizing carrier whether it is ceramic or not, filling and removal of the immobilizing carrier should not prevent the cells from achieving their uniform adhesion onto said immobilizing carrier.
With this method, however, a quantity of cell suspension supplied to the immobilizing carrier stack is initially apt to stagnate at the cell suspension supply side on the stack surface and at the area adjacent the supply pipe. This is inconvenient in that a long time is taken before the cells can be proliferated through out the whole immobilized carrier stack.
The cell culture apparatus of the present invention intends to solve such problem.